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Published on: March 21, 2018
Analysis of c-Ki-ras mutations in human colon carcinoma by cell sorting, polymerase chain reaction, and DNA
G C Burmer1, P S Rabinovitch, L A Loeb
1Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington, School of Medicine, Seattle 98195.
Abstract:
We have analyzed colon carcinomas by a combination of histological enrichment, cell sorting, polymerase chain reaction, and direct sequencing of the c-Ki-ras-2 gene. DNA was chemically extracted from 50-microns sections of paraffin-embedded colon carcinomas and amplified in vitro, and mutations were documented directly by DNA sequencing. Enrichment for tumor cells was obtained histologically and by sorting nuclei on the basis of DNA content differences. Mutations in codon 12 were present in both aneuploid and diploid subpopulations of sorted carcinomas, suggesting that these mutations precede ploidy alterations in the progression of these neoplasms. We have demonstrated the feasibility of utilizing DNA from tissues treated with different fixatives, including methyl carnoys, formalin, and Hollande's solution. This procedure allows one to retrospectively reconstruct the temporal relationship between the occurrence of mutations and sequential morphological changes during tumorigenic progression.
Insights
Colon carcinoma analysis reveals c-Ki-ras-2 gene mutations in codon 12. These mutations appear to occur before changes in DNA content during tumor progression.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Colon carcinomas are complex neoplasms with genetic alterations.
- Understanding the sequence of genetic and ploidy changes is crucial for cancer progression research.
Purpose of the Study:
- To investigate the temporal relationship between c-Ki-ras-2 gene mutations and ploidy alterations in colon carcinomas.
- To establish a method for analyzing DNA from archival paraffin-embedded tissues.
Main Methods:
- Histological enrichment and cell sorting for tumor cell isolation.
- Polymerase chain reaction (PCR) and direct DNA sequencing of the c-Ki-ras-2 gene.
- Analysis of DNA from paraffin-embedded colon carcinoma sections fixed with various agents.
Main Results:
- Mutations in codon 12 of the c-Ki-ras-2 gene were identified in both diploid and aneuploid tumor cell populations.
- The presence of these mutations in both subpopulations suggests they precede ploidy alterations.
- Demonstrated feasibility of DNA analysis from tissues preserved with common fixatives (methyl carnoy's, formalin, Hollande's solution).
Conclusions:
- c-Ki-ras-2 gene mutations at codon 12 likely occur early in colon tumorigenesis, before significant changes in DNA content.
- The developed methodology enables retrospective analysis of genetic mutations and morphological changes in archival cancer tissues.
- This approach facilitates a deeper understanding of the molecular events driving tumor progression.
